James McElroy, Oscar Salas-Solano, Lynn Gennaro, Genentech, Inc.,
South San Francisco, CA USA
CE-SDS has emerged as a powerful methodology in the biopharmaceutical
industry to support analytical characterization, process development,
and quality control of therapeutic monoclonal antibodies (rMAbs).
Typically, UV absorbance is a common detector in CE-SDS, however, the low
sensitivity (equivalent to Coomassie stained gels) has limited its
application in the biotechnology industry. At Genentech Inc., we demonstrated
that precolumn labeling of rMAbs with 5-TAMRA.SE and LIF detection can
be used for CE-SDS analyses as a replacement for silver-stained
SDS-PAGE to quantify low-level impurities and rMAb size variants and to
determine lot-to-lot consistency. This method requires the use of manual
dye-labeling protocols. The protein sample must be buffer-exchanged to
provide the optimal conditions for fluorescence dye labeling followed by
excess dye removal purification. This sample preparation procedure can
often be the bottleneck in sample throughput, and automated procedu
res are desirable. This poster describes an automated sample
preparation protocol for CE-SDS-LIF using a liquid handling robot. This system
allows the automation of the dye labeling protocol as well as
decreasing the amount of protein sample required by ten fold. This greatly
increases throughput while reducing analyst sample handling and quantity of
sample used.
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